Proteins are a medically relevant class of substances which are involved in many important processes in the body, e.g., as enzymes, as means for transporting other molecules, and as clotting factors in the blood. It is particularly useful, especially in the case of disease, to detect such proteins or the quantities or concentrations thereof, for example, in the blood. Chemical, enzymatic, optical, and other methods of diagnostic testing are used for this purpose. Proteins have a three-dimensional structure, which is defined by their amino acid sequence and used by the immune system, through specific antibody recognition, wherein antibodies are able to distinguish between foreign proteins and their own proteins. This interaction between the antibody and the so-called antigen can be used as an immunological assay, and has become the established standard method used in in vitro diagnostics.
This principle is also applied in a “competition assay” to determine the concentration of an antigen. In this case, at the start of the test the antibodies are provided with antagonists that bind to the antibody, for example, wherein the antagonists bear a fluorescent labeling, whereby an optical measurement signal is generated. In the presence of the substance to be detected—an analyte—some of the fluorescent labeled antagonists are displaced from the binding site of the antibody by the analyte. As a result, the number of antibodies having fluorescent labeling is decreased, and therefore, a smaller measurement signal is derived. This simplest form of competition assay involves a purified or recombinantly produced labeled antigen, which is present together with the binding antibody as an antigen/antibody complex. The unlabeled antigen (analyte) to be measured binds to the antibody with the same affinity and replaces the labeled molecule to a certain extent. If the equilibrium constant of the reaction is known, the concentration of unmarked antigen can be to determined from the measurement of the replacement.
Also known are in vitro methods for molecule detection without optically measurable labeling. In this case, field effect transistors (FET) are used in vitro, for example. These approaches involve the molecule to be detected reaching a sensitive layer of the FET as a result of diffusion. For this to occur, the molecule must carry a charge in order for the charge transfer that is necessary for detection to take place. Alternatively, catcher molecules (e.g., antibodies) can be immobilized on the sensitive layer of the FET, which molecules are able to recognize, bind to, and concentrate specifically the analyte to be detected. The binding of the analyte to the antibody layer results in the charge transfer.
Over the past several decades, it has been found that an in vitro assay of analytes is often insufficient for a reliable determination of a current and relevant status of the analyte, and is thus insufficient for a reliable determination of a condition of a patient. Rapid intervention is essential, particularly in the event of acute changes in chronically ill patients, for example. In such cases, continuous monitoring of analytes and concentrations thereof over months or even years is recommended. Therefore, a sensor system is needed which is capable of reliably and rapidly monitoring analytes, for example, in vivo over an extended period of time.
The currently known sensor systems are unsuitable for in vivo applications. An antibody or competition assay, as described above, can ordinarily be used only a single time in a detection process because the antibodies bind so securely to their specific antigen, and the formed bond cannot be easily separated. An assay of this type does provide a highly precise, but single measurement of the concentration of the substance during one moment in time. However, in order to monitor a concentration of analyte over a period of months/years it is necessary for the bond between antibody and analyte to be reversible, so that in addition to providing a single instantaneous measurement, the assay can also detect varying concentrations. Moreover, the antibodies used for this purpose are not particularly stable under long term contact with bodily fluids.
In addition, most optically active labels are hazardous to a patient's health or even toxic, particularly if they escape from the sensor system (for example, as a result of decomposition processes) and enter the body, where they can evoke critical reactions and/or inflammatory reactions. This also results in a depletion of detection molecules and a deactivation of the optically active labels, for example, as a result of photobleaching, thereby impairing the functionality of the sensor system. In addition, for antibody detection methods, several washing steps are typically required for eliminating unbonded or interfering molecules. For an implantable sensor having an assay as described above, this would require a microfluid system and therefore a significant enlargement of the implant (including a rinsing reservoir), which would be unreasonable for implantation into a patient.
The molecules that are typically detected using FET's are highly charged polyelectrolytes, such as DNA, for example. However, under physiological conditions most proteins carry a very low charge and cannot routinely be detected using an FET unless special labels, such as nanoparticles, which often pose a health hazard, are used to amplify the signal.
Heretofore, only pH sensors and blood gas sensors (electrodes) have become established as reliable electrical methods for detecting a condition of the blood. These sensors are used mostly in intensive care units, in which case continuous access to the patient's blood via shunts and catheters is required. A sensor system of this type is not suitable for continuous use over a period of months or years.
Therefore, there is no currently known fully implantable sensor system that will remain stable over the long term, and which is capable of monitoring and determining the concentration of larger analytes (such as proteins) over a period of months or even years. No approaches are known which describe a fully implantable detection system for proteins on the basis of antibodies. Heretofore, only in vitro systems which cannot be used in vivo are known.